Transcriptional repression is an important feature of developmental processes where it is necessary for establishing complex patterns of gene expression. The broad long-term goal of the proposed study is to determine the precise molecular mechanism(s) used by developmental transcriptional repressors and the roles played by their associated cofactors. We are particularly interested in the Hairy/Enhancer of split/Deadpan (HES) family of basic-Helix-Loop-Helix (bHLH) proteins that function as dedicated repressors important in diverse developmental processes including segmentation, neurogenesis, myogenesis, somitogenesis, and, when mis-regulated, the onset of leukemias/cancers. Hairy is a HES family transcriptional repressor that sits at a key position in the segmentation gene hierarchy: it is one of the first genes to show overt reiterated periodicity that is central to the establishment of proper embryonic body plan throughout metazoa. This study will use genetic, developmental, molecular, biochemical, and functional genomic approaches to further characterize the Hairy developmental repressor and to determine the rules governing its cofactor recruitment. The specific aims of this proposal are to identify and characterize Hairy's direct targets during embryonic segmentation and to investigate the mechanism(s) governing the context-dependent nature of cofactor recruitment and Hairy-mediated transcriptional repression. bHLH proteins are often involved in developmental "switch" decisions: they regulate the choice between alternate pathways and effect changes in cell fate. Mistakes in these regulatory steps often lead to developmental defects and the onset of cancers. The numerous processes requiring bHLH proteins and the wealth of techniques/reagents available in Drosophila make it an excellent organism for studying the functions of these proteins in vivo. Results obtained from these projects are expected to have wide implications as HES family proteins and mechanisms of transcriptional repression are conserved throughout organisms.